There are a number of monogenetic diseases, including genetic mutations that introduce a premature stop codon into a gene, resulting in either no gene product or in a gene product made by expressing the gene that is either wholly or partially inactive. Such diseases include, for example, Duchenne Muscular Dystrophy (DMD), Cystic Fibrosis (CF), and may well include some genetic mutations which lead to the development of various cancers.
Duchenne Muscular Dystrophy (DMD) is due to the mutation of a gene in the X chromosome coding for a protein called dystrophin. The mutations of the dystrophin protein vary from one family of patients to another, but it always results in the absence of a functional dystrophin protein under the membrane on the muscle fiber. The absence of the dystrophin protein increases the vulnerability of the muscle fibers during contraction. Accordingly, repeated cycles of contraction and relaxation of the muscle produces a progressive reduction of the number of muscle fibers. The end result is a loss of strength which confines many patients to a wheel chair by the age of ten and in many cases to premature death in their early twenties.
Roughly 70% of the mutations of the dystrophin gene are large deletions of one of several exons, Still other mutations are small point mutations due either to a small deletion of a few base pairs leading to a shift of the reading frame, or changes of only one base pair producing a missense or a stop codon within the gene. It is estimated that about 5% of all DMD mutations may be due to premature stop codons in the gene.
Cystic fibrosis (CF) is due to a mutation of a gene coding for the CF transmembrane conductance regulator (CFTR) protein. Experiments with a bronchial epithelial cell line obtained from a CF patient having a premature stop mutation in the CFTR gene having confirmed this hypothesis. This mutation results in a premature end of the synthesis of the CFTR protein and thus is a non-functional protein. Early attempts to address this condition included treating these cells with aminoglycoside antibiotics G418 (100 mg/ml) or with gentamicin (200 mg/ml) for about 18 to 24 hours. Incubation with gentamicin suppressed the premature stop mutation by enabling the ribosome to insert an amino acid at the premature stop codon. Accordingly, a full-length CFTR protein is produced. The suppression of the premature stop codon by gentamicin is thought to be mediated by mis-pairing between the stop codon and a near-cognate aminoacyl tRNA. Furthermore, work has demonstrated that the full length CFTR protein resulting from the incubation with the aminoglycoside antibiotics is present in the cell membrane and is functional.
The mdx mouse is an animal model for DMD. The mouse's gene includes a point mutation in the dystrophin gene resulting in a truncated protein which is not incorporated in the muscle fiber membrane. Accordingly, this animal model presents an opportunity to test the effect of various compounds that promote readthrough of premature stop codons for their effect on masking the defect. For additional reading on the causes of some forms of DMD and cystic fibrosis and attempts to treat these diseases using gentamicin, please see U.S. Pat. No. 6,475,993, which is incorporated by reference herein in its entirety.
One strategy put forward to treating diseases of this nature is to provide a medicant that enables readthrough of premature stop codons; One such class of drugs with this capacity is the widely-used antibiotics referred to as aminoglycosides. While these drugs may have the potential to provide treatment for such disorders, they have a serious problem in that many, if not all of them, including the natural occurring form of gentamicin exhibit severe nephrotoxicity and/or ototoxicity. Accordingly, most naturally-occurring aminoglycosides cannot be prescribed on a daily basis for the lifetime of the patient without heightening the risk of acute kidney failure or inner ear toxicity.
Given the devastating effects that these various diseases have on the individuals afflicted with them, and the lack of treatments available for these diseases, there is a compelling need for various methods and materials which can be used to treat these diseases. Various aspects of the following disclose detail materials and methods for addressing this need.